SAFRANINE-O AS MEMBRANE-POTENTIAL PROBE - A MECHANISTIC STUDY USING FLUORESCENCE SPECTROSCOPY

Use of safranine-o has been examined as membrane potential probe in 1- palmitoyl-2-oleoyl-3-phosphatidylcholine (POPC) vesicles both in prese nce and absence of cholesterol. The fluorescence signal increases in p resence of vesicles and the increase in fluorescence intensity on hype rpolarization with valinomycin is diffusion potential dependent. The f luorescence spectra recorded after time driven experiments reveals the blue shift in lambda(max) of fluorescence with increasing diffusion p otential. The fluorescence spectra of vesicles-associated dye is at va riance with those of the safranine-o in organic solvents. In organic s olvents with increasing hydrophobic character of the solvent the lambd a(max) is slightly red shifted. The electronic spectra of the dye mole cule and the charges on different atomic centers have been calculated by quantum chemical method GRINDOL. The predicted first excited state originating from the phenazine moiety is in very good agreement with t he excitation wavelength. On the basis of charges on various atoms the binding of safranine with vesicles has been discussed. The nonlinear behaviour of fluorescence signal with Delta phi, anisotropy measuremen ts and the computational results, reveal the penetration of bound dye molecules (along with orientation) as a function of diffusion potentia l. Addition of microaliquots of 1.5 M K2SO4 to already hyperpolarized vesicles decreases the fluorescence signal and the fluorescence spectr a recorded on stabilization of signal after each addition showed a shi ft in lambda(max) of fluorescence in opposite direction i.e. red shift ed.